Hypertonicity (e.g. high NaCl) activates the esential osmoprotective transcription factor NFAT5 by increasing its abundance, nuclear localization and transactivating activity. It is activated by a network of signaling molecules, whose members we have continued to identify and characterize, as follows: Many high-NaCl-induced perturbations and protective responses are known, but the signaling pathways involved are less clear. Change in protein phosphorylation is a common mode of cell signaling, but there was no unbiased survey of protein phosphorylation in response to high NaCl. We used stable isotopic labeling of amino acids in cell culture coupled to mass spectrometry to identify changes in protein phosphorylation in human embryonic kidney (HEK 293) cells exposed to high NaCl. We reproducibly identify >8,000 unique phosphopeptides in 4 biological replicate samples with a 1% false discovery rate. High NaCl significantly changed phosphorylation of 253 proteins. Western analysis and targeted ion selection mass spectrometry confirm a representative sample of the phosphorylation events. We analyzed the affected proteins by functional category to infer how altered protein phosphorylation might signal cellular responses to high NaCl, including alteration of cell cycle, cyto/nucleoskeletal organization, DNA double-strand breaks, transcription, proteostasis, metabolism of mRNA, and cell death. Having previously found that high NaCl causes rapid exit of 14-3-3 isoforms from the nucleus, we used siRNA-mediated knockdown to test whether 14-3-3s contribute to the high NaCl-induced increase in the activity of NFAT5. We found that, when NaCl is elevated, knockdown of 14-3-3-&#946; and/or 14-3-3-&#949; decreases NFAT5 transcriptional activity, as assayed both by luciferase reporter and by the mRNA abundance of the NFAT5 target genes aldose reductase and the sodium- and chloride-dependent betaine transporter, BGT1. Knockdown of other 14-3-3 isoforms does not significantly affect NFAT5 activity. 14-3-3-&#946; and/or 14-3-3-&#949; do not act by affecting the nuclear localization of NFAT5, but by at least two other mechanisms: (1) 14-3-3-&#946; and 14-3-3-&#949; increase protein abundance of NFAT5 and (2) they increase NFAT5 transactivating activity. When NaCl is elevated, knockdown of 14-3-3-&#946; and/or 14-3-3-&#949; reduces the protein abundance of NFAT5, as measured by Western blot, without affecting the level of NFAT5 mRNA, and the knockdown also decreases NFAT5 transactivating activity, as measured by luciferase reporter. The 14-3-3s increase NFAT5 protein, not by increasing its translation, but by decreasing the rate at which it is degraded, as measured by cycloheximide chase. It is not clear at this point whether the 14-3-3s affect NFAT5 directly or indirectly through their effects on other proteins that signal activation of NFAT5. Several studies pointed to a possible connection between nuclear translocation and DNA binding of NFAT5; however, the mechanism of NFAT5 nuclear translocation and the effect of DNA binding on retaining NFAT5 in the nucleus were largely unknown. Recent experiments showed that different mutations introduced in the DNA-binding loop and dimerization interface were important for DNA binding and some of them decreased the nuclear-cytoplasm ratio of NFAT5. To understand the mechanisms of these mutations, we modeled their effect on protein dynamics and DNA binding. We showed that the NFAT5 complex without DNA is much more flexible than the complex with DNA. Moreover, DNA binding considerably stabilizes the overall dimeric complex and the NFAT5 dimer is only marginally stable in the absence of DNA. Two sets of NFAT5 mutations from the same DNA-binding loop were found to have different mechanisms of specific and nonspecific binding to DNA. The R217A/E223A/R226A (R293A/E299A/R302A using isoform c numbering) mutant is characterized by significantly compromised binding to DNA and higher complex flexibility. On the contrary, the T222D (T298D in isoform c) mutation, a potential phosphomimetic mutation, makes the overall complex more rigid and does not significantly affect the DNA binding. Therefore, the reduced nuclear-cytoplasm ratio of NFAT5 can be attributed to reduced binding to DNA for the triple mutant, while the T222D mutant suggests an additional mechanism at work.